NIH Public Access Author Manuscript Hypertension. Author manuscript; available in PMC 2013 August 01. Published in final edited form as: Hypertension. 2012 August ; 60(2): 459–466. doi:10.1161/HYPERTENSIONAHA.112.191270. $watermark-text Chronic Matrix Metalloproteinase Inhibition Retards Ageassociated Arterial Proinflammation and Increase in Blood Pressure Mingyi Wang*,#, Jing Zhang#, Richard Telljohann, Liqun Jiang, James Wu, Robert E. Monticone, Kapil Kapoor, Mark Talan, and Edward G. Lakatta Laboratory of Cardiovascular Science, Intramural Research Program, NIA/NIH, Baltimore, MD 21224 Abstract $watermark-text Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, and an increase in arterial pressure. This arterial remodeling is linked to proinflammatory signaling, including transforming growth factor-beta1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1), and proendothelin-1(Pro-ET-1), activated by extracellular matrix metalloproteinases (MMPs) and orchestrated, in part, by the transcriptional factor ets-1. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-mo-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of MCP-1 and TGF-β1 activation; (5) a diminution in the activity of the pro-fibrogenic signaling molecule, SMAD-2/3 phosphorylation; (6) inhibition of pro-ET-1 activation; and (7) down-regulation of expression of ets-1. Acute exposure of cultured vascular smooth muscle cells (VSMC) in vitro to pro-ET-1 increased both the transcription and translation of ets-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of VSMC with an adenovirus harboring a full-length ets-1 cDNA increased activities of both TGF-β1 and MCP-1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by perseveration of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure. $watermark-text Keywords Aging; Arterial remodeling; Matrix metalloproteinase inhibitor; Endothelin-1; Transforming growth factor-beta 1; ets-1; Monocyte chemoattractant protein-1; Blood pressure * Address for correspondence: Mingyi Wang M.D, Ph.D., Staff Scientist, Laboratory of Cardiovascular Science, 5600 Nathan Shock Drive, National Institute on Aging-National Institutes of Health, Baltimore, Maryland 21030, United States of America, Phone: 410-558-8112, Fax: 410-558-8150, mingyiw@grc.nia.nih.gov. #Equal contribution Competing interest The broad-spectrum MMP inhibitor PD166793 was generously provided by Global Research & Development, Pfizer Inc. (Groton, CT). Wang et al. Page 2 Introduction $watermark-text Advancing age is a major risk factor for hypertensive and atherosclerotic complications due, in part, to an increase in local arterial wall inflammatory signaling that is linked to an enhanced activity of extracellular matrix metalloproteinases (MMPs) [1–5]. MMPs are a family of zinc-dependent endopeptidases that includes gelatinases MMP-2/-9, collagenases MMP-7/-13, and matrilysin MMP-7 [6]. In general, these proteinases are capable of degrading numerous extracellular matrix proteins, but also can process a number of bioactive molecules, which tightly control the turnover of matrix [6–12]. MMP gene polymorphism is closely associated with increases in arterial calcification and stiffness in humans with hypertension and atherosclerosis [13, 14]. $watermark-text MMP mRNA, protein, and activity are markedly increased in the aged arterial wall of rodents, nonhuman primates, and humans [6, 11, 15, 16]. MMPs trigger a local proinflammatory signaling loop that disrupts arterial extracellular structure and causes vasoconstriction [4, 12, 14–18]. Matrix metalloproteinase type II (MMP-2) relays signaling of monocyte chemoattractant protein-1 (MCP-1) and triggers transforming growth factorbeta 1 (TGF-β1) activation, which, in a feed forward manner, activates both MMP-2 and MCP-1 [4]. Canonically, activation of this signaling loop with aging not only results in increased cellularity and thickening of the arterial intima, but also causes elastin network fracture, the release of soluble fibrillin-1, and collagen deposition in the arterial wall [4, 7, 11]. Noncanonically, however, MMP catalytic action, resembles that of endothelin converting enzyme (ECE), enhancing the conversion from the “big” inactive proendothelin-1 (pro-ET-1, 1–31) to the “small” active vasoconstrictor endothelin-1 (ET-1, 1– 21) [16]; This effect synergizes with other MMP effects to activate proinflammatory pathways and to increase arterial constriction [15, 16]. The MMP-related extracellular local proinflammatory signaling cascade is orchestrated, in part, by the nuclear transcriptional factor ets-1 [17, 18]. Importantly, ets-1 is an early response gene to ET-1 receptor activation [19]. $watermark-text We hypothesized that chronically moderating the influence of MMP activation by MMP inhibition would retard arterial proinflammatory signaling and reduce its age-associated pathophysiological consequences. Indeed, in the present study, chronic administration of an MMP inhibitor, PD 166793, to 16-mo-old rats for 8 months compared to placebo substantially reduces the activation of gelatinases and interstitial collagenases within the arterial wall; preserves the intact network of elastin; blunts TGF-β1 and MCP-1 activation, and alleviates collagen deposition; and considerably blocks the conversion of the inactive pro-ET-1 to its active form; reduces expression of its early response gene ets-1; and markedly blunts an age-associated increase in blood pressure. These age-associated features of arterial wall proinflammation and effects of MMP inhibition in vivo are recapitulated early passage vascular smooth muscle cells (VSMC) in vitro. Thus, MMP inhibition is a novel therapeutic approach to retard age-associated proinflammation and its pathophysiologic consequences. Materials and Methods Experimental animals and treatment The current study was implemented in male Fisher 344 crossbred Brown Norway rats (F344XBN). This strain exhibits age – associated central arterial remodeling including collagen deposition and elastin degradation and develops moderate hypertension between age 8 and 30 mo, which is closely associated with their increased mortality [7, 11, 20]. Furthermore, administration of PD166793(5 mg/kg/d), an MMP inhibitor, to rats for 4 months produces a plasma drug level of 100 μmol/, which abolishes the activity of MMP-2, Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 3 $watermark-text -9, and -13 [21]. To avoid confounding the growth phase of the arterial wall and the potential deleterious effects of growth modulation of MMP activity as well as a high mortality and to determine the effects of MMP inhibition on the age-associated increase in blood pressure and arterial phenotypes, a treatment group (n=15) of 16-month-old male rats received daily administration of a broad-spectrum MMP inhibitor, PD166793, administrated by gavage (5mg/kg/day, Global Research & Development, Pfizer Inc., Groton, CT) in 0.1 % dimethyl sulfoxide (DMSO); for 8 months served as 24 moth-old inhibitor treatment group (14Mi) a placebo group (n=15) of 16-month-old male rats received daily administration of the same volume of 0.1 % DMSO, continued for 8 months as 24 month-old placebo group (24M); and an untreated young reference group (n=15) of 8-month-old male rats was administrated with the same volume of 0.1 % DMSO for 8 months as 16-month-old reference group (16M). Notably, three rats in the placebo group, and two rats in the PD166793 treated group died during the trial period. The cause of death was unknown, and these rats are not included in the analysis. All procedures were performed according to protocol (341-LCS-2007) approved by the Institutional Animal Care and Use Committee and complied with the guide for the care and use of laboratory animals (NIH publication No. 3040-2, revised 1999). See online supplement for details of other materials and methods: Blood pressure $watermark-text measurement; Tissue harvesting; Histology; Immunohistochemistry and immunofluorescence; Polyacrylamide gel electrophoresis and in situ zymographies and MMP-13 activity assay; Vascular smooth muscle cell isolation, culture, and treatment; Generation of recombinant adenoviruses and vascular smooth muscle cell infection; Quantitative real-time PCR; Western blot analysis; and Statistical analyses. Results MMP inhibition effectively reduces age-associated increases in aortic gelatinase and interstitial collagenase activity in situ $watermark-text In vitro polyacrylamide gel electrophoresis (PAGE) gelatin zymographs to detect gelatinase activity of rat aorta (Figure S1A) show that the re-natured gelatinase, MMP-2, is increased with aging, while the re-natured matrix metalloproteinase type 9 (MMP-9) is rarely detected. Importantly, gelatin zymographs of the arterial wall in situ indicate that aortic gelatinase activity (green color) increases with aging (Figure 1A), consistent with previous reports [11]. In situ aortic gelatinase activity is markedly decreased by chronic treatment with PD 166793 (24Mi) group compared to the placebo group (24M) (Figure 1A). PAGE casein zymography (Figure S1B), which was employed to detect activity of collagenases MMP-1, MMP-13 and matrilysin MMP-7 based on levels of their molecular weights [22], shows that the re-natured arterial MMP-13 activity increases with aging (the lowest weak bands), which is consistent with the level of its protein expression (Figure S1C). Importantly, an MMP-13 antibody-capture assay indicates that PD166793 treatment markedly reduces aortic MMP-13 activity in 24M compared to 24Mi group rats (Figure S1D). Casein zymograms in situ confirm that the capacity to digest casein (red color) increases with aging (Figure 1B), and also shows that age-associated enhanced digestive capability is markedly inhibited in 24Mi compared to the 24M group (Figure 1B). In addition, collagen zymograms in situ further show that the capacity to digest collagen increases with aging, in particular in the thickened intima (green color, Figure 1C), and also shows that age- Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 4 associated enhanced digestive capability is markedly inhibited in 24Mi compared to the 24M group (Figure 1C). MMP inhibition reduces age-associated aortic extracellular matrix remodeling $watermark-text Morphologic analysis (Table S1) indicates that intimal thickness (IT), medial thickness (MT), and intimal medial thickness (IMT) significantly increase with age from 16- to 24-mo (24M vs.16M). Although the age-associated increase in IT, MT, and IMT is not substantially reduced, histochemical staining and morphometric analysis of the elastin fraction/density reveal that age-associated degradation (decreased elastin density) in the arterial wall are reduced in 24Mi vs. 24M (Figure 2A). Furthermore, Western blot analysis demonstrates the formation and release of the elastin microfibril-apparatus breakdown product, soluble fibrillin-1, is completely abolished in 24Mi (Figure 2B) Collagen I immunolabelling shows that aortic collagen type I deposition, which increases with age, is prevented in 24Mi (Figure 2C). This finding is further confirmed by Western blot analysis (Figure 2C). MMP inhibition reduces age-associated aortic inflammation $watermark-text It is known that the proinflamatory MCP-1/TGF-β1 signaling loop is involved in arterial collagen disorders with aging [4, 7, 8, 11]. Western blot analysis (Figure 3A) shows that the active dimer form of MCP-1 [23, 24] is increased in the arterial wall with advancing age, and is markedly decreased in 24Mi. Immunostaining demonstrates that activated TGF-β1 is increased in the arterial wall with advancing age, particularly in the thickened intima, and this is markedly decreased in 24Mi (Figure 3B). Western blot analysis further confirms these findings (Figure 3C). Immunostaining and Western blot analyses show that the amount of activated p-SMAD2/3 and the number of stained VSMC nuclei for p-SMAD2/3, effective downstream signaling molecules of the TGF-β1 cascade, are also markedly increased with aging, and these increases are significantly reduced by MMP inhibition (Figure 3D). MMP inhibition modifies post-translational processing of arterial vasoconstrictor ET-1 Prior in vitro studies [16] document that MMP-2 has a potent capacity to cleaved latent “big” pro-ET-1 to the “small” form or activated ET-1, a much more effective vasoconstrictor than the precursor [15,16]. Figure 4A shows that arterial small ET-1 protein expression is up-regulated with aging, particularly within the intima and innermost media, confirming a recent report [15]. Chronic MMP inhibition significantly decreases small ET-1 (Figure 4B). Note that, that the small active form of ET-1 is undetectable in 16-mo-rat aorta. $watermark-text MMP Inhibition reduces expression of the proinflammatory transcription factor ets-1 A growing body of evidence indicates that ET-1 signaling and its effects to increase collagen production via TGF-β1 and MCP-1 upregulation are orchestrated by the transcription factor, ets-1 [17–19]. Immunostaining shows that the number of ets-1 stained VSMC nuclei (activated form) within the aortic wall in vivo is markedly increased in the 24M compared to the 16M group (Figure 4C). This age effect is abolished by MMP inhibition (Figure 4C, right panel). Western blot analysis further confirms this finding (Figure 4D). MMP inhibition blocks the ET-1/ets-1 signaling in aortic VSMC in vitro Previous studies demonstrate that MMP-2 enhances expression of inflammatory signaling molecules TGF-β1 and MCP-1 [4, 7], which is orchestrated, in part, by the nuclear transcriptional factor ets-1 in VSMC [17, 18]. We next explore whether MMP inhibition blocks the ET-1/ets-1 proinflammatory signaling in aortic VSMC in vitro. In early passage aortic VSMC expression of ets-1 protein is increased in a dose-dependent manner by treatment with active ET-1 (Figure 5A). Importantly, the ability of pro-ET-1 to increase both Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 5 ets-1 transcription and its translation is also markedly reduced by MMP inhibition (Figure 5B & C). To demonstrate a role of ets-1 in arterial proinflammatory signaling, we overexpressed ets-1 in VSMC via an adenovirus harboring a full-length ets-1 cDNA (Figure 5D, upper panel). Overexpression of ets-1 substantially increases both active MCP-1 and TGFβ1(Figure 5D, middle panels). The age-associated increase in arterial pressure is lowered by MMP inhibition $watermark-text An increase in arterial pressure is an important functional readout of age-associated arterial wall proinflammation [1–3, 25]. Figure 6 demonstrates that the anti-inflammatory effects of MMP inhibition (Figures 1–5) are accompanied by a marked blunting of the increase in blood pressure that occurs between 16 and 24 mo in this rat strain. Other effects of MMP inhibition MMP inhibition did not affect either diet or body weight of rats compared to placebo group (Table S2). Notably, MMP inhibition similarly diminishes age-associated coronary extracellular matrix remodeling and proinflammation (Table S1 & Figure S2). Discussion $watermark-text MMP activation is an element within an age-associated proinflammatory signaling circuit. Active MMP functions as an effective molecular scissor with broad structural/functional consequences (Figure S3). Our results demonstrate, for the first time, that PD 166793, a broad spectrum MMP inhibitor, retards a local inflammatory signaling loop, alleviates adverse extracellular matrix remodeling in both the aortic and coronary arterial walls, and that these effects are accompanied by a blunting of the age-associated increase in blood pressure. $watermark-text PD 166793 in the 10–100 μM range has global MMP inhibitory activity that having high affinity for MMP-2 and -13, and lower affinity for MMP-1, -7 and -9 [21, 26–28]. Note that PD 166793 does not exhibit inhibitory activity of other proteases, e.g., angiotensin converting enzyme, endothelin converting enzyme or tissue necrosis factor-alpha convertase [21, 29, 30]. A 5-mg/kg dose of PD 166793 was selected for our study because this dose produces plasma drug levels of 100 μM after 4 months and markedly reduces the aforementioned MMP activities in rats [21]. Indeed, the present findings show that ingestion of this dose after 4 months markedly retards an age-associated increase in blood pressure in rats and inhibits activity of aortic gelatinases and collagenases in situ. Our results show that the activities of both MMP-2 and MMP-13 increase within the aortic wall with aging. It is known that activated MMP-2 binds to elastin fibers and increases the cleavage of elastin, resulting in the dissolution of the structural microfibril-associated apparatus and the release of fibrillin-1 [5, 7, 8, 10, 31]. Activated MMP-2 also binds to the basement membrane of VSMC enabling their migration and to the basement membrane of endothelial cells enabling their desquamation [4, 9, 31]. Activated MMP-13 attaches to and cleaves intact types I or III collagen results in a release of collagen and length fragments [32–34]. These fragments are further cleaved into growth factor-like, “matrikines”, which activate MMPs in a feed forward manner [35, 36]. MMP inhibition maintains the intact scaffold of the aorta via reduction of the activity of gelatinase and interstitial collagenase (Figure S3). MMPs not only cleave elastin and collagen fibers, but also cleave latent TGF-β1, releasing soluble LTBP-1, to initiate the processes of TGF-β1 activation, which results in SMAD2/3 phosphorylation and production of collagen in VSMC [4, 7, 37]. The present study confirms Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 6 and extends previous findings that MMP increases pro-ET-1 activity, enhances ets-1 activation, and that, in turn, increases both MCP-1 and TGF-β1 activation, reinforcing their downstream molecule SMAD-2/3 phosphorylation and collagen production in VSMC [4, 15, 16]. MMP inhibition also diminishes the age-associated increase in arterial fibrosis via intervening on the proinflammatory signaling loop. Our findings suggest that MMP inhibition restores a balance of collagen production and cleavage within the arterial wall, resulting in a retardation of age-associated arterial fibrosis. Importantly, Figure S3 implicates each of these cleaved products generated by MMPs as an element of the proinflammation circuitry of either the Ang II or ET-1 cascade within the arterial wall with aging. $watermark-text $watermark-text An age-associated increase in arterial pressure is a clinical hallmark of aging [25], and results from joint effects of multiple factors, including, intimal-medial thickening, arterial proinflammatory responses, and vasoconstrictor of angiotensin II (Ang II) and ET-1 effects [1–3, 38, 39]. The components of the renin angiotensin aldosterone system, including angiotensinogen, angiotensin-converting enzyme, angiotensin II, its receptor AT1, and aldosterone protein and signaling are increased in the aged arterial wall [5, 8–10, 40–43]. So too, the present study, as well as others show that ET-1 protein and its activity are increased in the arterial wall with aging [15, 42, 43 ]. MMP inhibition reduces the age-associated increase in arterial blood pressure likely, in part, via blockade of the ET-1 and Ang IIassociated proinflammatory signaling loop and matrix remodeling. These effects contribute to decreased vasoconstriction and blood pressure, even without having significant effects on the intimal-medial thickening. In addition, PD166793 reduces production and activity of cardiovascular reactive oxygen species (ROS), which also modulate blood pressure [44, 45]. Angiotensin II (Ang II) enhances ET-1 expression in the arterial wall [46]. Blockade of the renin-angiotensin aldosterone system signaling, including angiotensin converting enzyme (ACE inhibitor), angiotensin II receptor 1 (AT1) antagonists, and aldosterone blockers, all are capable of retarding age-associated arterial disorders, including increases in intimalmedial thickness, arterial stiffness, and blood pressure [47–49]. Inhibition of ET-1 also alleviates endothelial dysfunction and arterial stiffness with aging and hypertension, in part, via inhibition of MMP-2 activation [50, 51]. In addition, inhibition of ROS production/TGFβ1/MMP2 activation, by deletion of the gene p66Shc, nitrite supplementation, or exercise, substantially improves age-associated endothelial dysfunctions and arterial stiffening [52– 55]. $watermark-text Perspectives MMP activation in age-associated arterial remodeling is a convergence point of multiple inflammatory stress pathways including Ang II, ET-1, and mechanical forces. Our unique study provides proof of concept that MMP inhibition can attenuate the extent and rate of adverse arterial remodeling that accompanies aging. Although the currently available MMP inhibitors have undesirable side effects, including the delay of wound healing and impairment of angiogenesis as well as skeletal muscle damage [56–58], advances in structurally adjustment of existing inhibitors to increase selectivity, remove toxicity and improve bioavailability in newer versions show promise. Thus, MMP inhibition may offer a future preferable therapeutic approach to maintain arterial health during aging. Molecular components and signaling networks of age-associated arterial remodeling are recaptured in young subjects with hypertension and atherosclerosis, and arterial aging is a chronic process intimately linked to subclinical arterial diseases including hypertension and atherosclerosis [2], Thus, MMP inhibition may also offer a potential therapeutic approach to retarding the development of this age-associated arterial diseases. Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 7 Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments Funding This research was supported by the Intramural Research Program of the National Institute on Aging. References $watermark-text $watermark-text $watermark-text 1. Lakatta EG, Wang M, Najjar SS. Arterial aging and subclinical arterial disease are fundamentally intertwined at macroscopic and molecular levels. Med Clin North Am. 2009; 93:583–604. [PubMed: 19427493] 2. 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Page 11 Novelty and Significance What Is New? • MMP inhibition prevents elastin degeneration, collagen deposition, and increase in arterial pressure associated with arterial ageing in rats by intervening on the MCP-1/TGF-β1/ET-1 proinflammatory signaling network. What Is Relevant? • $watermark-text Age-associated changes in proinflammation, vasoconstriction, elastin degeneration, and collagen deposition facilitate increases in both arterial stiffness and blood pressure in older persons Summary Our unique study provides proof of concept that MMP inhibition can attenuate the extent and rate of adverse arterial remodeling with aging. $watermark-text $watermark-text Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 12 $watermark-text Figure 1. MMP activation in situ. $watermark-text A) Fluorescence micrographs of in situ gelatin zymograms (green, 400X), and average intensity of the cleaved gelatin signal (lower panel, n=3/goup). B). Fluorescence micrographs of in situ casein zymograms (red, 400X) and average intensity of the cleaved casein signal (lower panel, n=3/group). C). Fluorescence micrographs of in situ collagenase zymograms (green, 200X) and the average intensity of the cleaved casein signal (lower panel, n=3/group). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media. $watermark-text Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 13 $watermark-text $watermark-text Figure 2. Aortic extracellular matrix remodeling A) Photomicrographs of elastic fibers (dark blue) in E.V.G. stained with elastin protein in aortic sections with (left panels, 400X). Average elastin fraction (EF, right panel, n=5/per group) B) Western blots of Fibrillin-1(left panels) and average data (n=3/group, right panel). C) Photomicrographs of Collagen I immunostaining (brown, 200X, left panels) of aortic sections, and average data (right panel, n=4/group). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media. $watermark-text Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 14 $watermark-text $watermark-text $watermark-text Figure 3. Proinflammatory signaling molecules A) Representative Western blots of the MCP-1 dimer (left panel), and average data (n=3/ group, right panel). B) Photomicrographs of aortic wall TGF-β1 staining (brown, 400X). C). Western blots of TGF-β1 (left panel), and average data (n=3/group, right panel). D) Photomicrographs of aortic wall p-SMAD2/3 staining (upper panels, brown color, 400X); and Western blots of p-SMAD2/3 (lower left panels), and average data (n=3/group, lower right panel). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media. Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 15 $watermark-text Figure 4. Endothelin-1 expression and cleavage, and transcriptional factor, ets-1, expression A) Immunolabelling of aortic wall ET-1 (brown color, 200X); B) Western blots of arterial wall ET-1 (left panel), and average data (right panel, n=3/group). #p< 0.05, 24M vs. 24Mi. C) Photomicrographs of aortic wall ets-1 staining (brown, 400X). D) Western blots of ets-1 (left panels), and average data (n=3/group). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media. $watermark-text $watermark-text Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 16 $watermark-text Figure 5. Proinflammation cascade within VSMC $watermark-text A) Western blots of ets-1 (left panels), and average data (right panel, n=3 independent experiments). * p<0.05, vs. control. B). RT-PCR analysis (n=4 independent experiments). vs control; #p<0.05, vs. PD166793 treatment. C) Representative Western blots of ets-1. D). Over-expression of ets-1 in VSMC increases activated MCP-1 and TGF-β1 protein. $watermark-text Hypertension. Author manuscript; available in PMC 2013 August 01. Wang et al. Page 17 $watermark-text $watermark-text Figure 6. Age-associated increases in systolic blood pressure (SBP) and diastolic blood pressure (DBP) are reduced by MMP inhibition $watermark-text The interaction between treatment groups and age on arterial pressure in linear mixed effects models is highly statistically significant for both SBP (*p = 0.0006); and DBP (#p = 0.0082). Hypertension. Author manuscript; available in PMC 2013 August 01.